EP1497440B1 - Stable adenoviral vectors and methods for propagation thereof - Google Patents

Stable adenoviral vectors and methods for propagation thereof Download PDF

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EP1497440B1
EP1497440B1 EP03753569A EP03753569A EP1497440B1 EP 1497440 B1 EP1497440 B1 EP 1497440B1 EP 03753569 A EP03753569 A EP 03753569A EP 03753569 A EP03753569 A EP 03753569A EP 1497440 B1 EP1497440 B1 EP 1497440B1
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pix
adenovirus
promoter
sequence
sequences
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EP1497440A2 (en
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Ronald Vogels
Menzo Jans Emco Havenga
David Adrianus Theodorus Zuijdgeest
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Janssen Vaccines and Prevention BV
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Crucell Holand BV
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • the invention relates to the field of medicine, more in particular the invention relates to recombinant adenoviral vectors and the use thereof.
  • Human adenoviruses are non-enveloped icosahedral particles of 60-90 nM size. To date 51 serotypes have been identified that are subdivided into 6 subgroups based on hemagglutination properties and sequence homology (Francki et al., 1991). The genome has a length of 34 to 36 kb and is flanked on both sites by inverted terminal repeat sequences (ITR). The virus infectious cycle is divided into an early and a late phase. In the early phase (6-8 hours after infection) the virus is uncoated and the genome transported to the nucleus after which the early gene regions E1-E4 become transcriptionally active.
  • ITR inverted terminal repeat sequences
  • the early region-1 contains two transcription regions named E1A and E1B.
  • the E1A region encodes two major proteins that are involved in modification of the host cell cycle and activation of the other viral transcription regions (reviewed by Russell, 2000).
  • the E1B region encodes two major proteins, 19K and 55K, that prevent, via different routes, the induction of apoptosis resulting from the activity of the E1A proteins (Rao et al.,1992; Yew and Berk, 1992; reviewed in Shenk, 1996).
  • the E1B-55K protein is required in the late phase for selective viral mRNA transport and inhibition of host protein expression (Pilder et al., 1986).
  • pIX An intermediate protein, pIX, is part of the capsid and is known to stabilize the hexon-hexon interactions (Furcinitti et al., 1989). In addition, pIX has been described to transactivate TATA containing promoters like the E1A promoter and MLP (Lutz et al., 1997).
  • adenoviruses Due to the extensive knowledge of the viral biology and the high efficiency of nuclear delivery after entry into cells, adenoviruses have become popular tools for gene delivery into human cells.
  • adenoviral vectors are stable and can be produced relatively easy at large scale. In most cases vectors are deleted for at least the E1 region, which renders them replication deficient. Production of E1-deleted vectors based on subgroup C serotypes Ad5 or Ad2 is achieved in E1 complementing cell lines such as 293 (Graham et al., 1970), 911 (Fallaux et al., 1996) and PER.C6 TM (Fallaux et al., 1998).
  • PER.C6 TM cells and matched adenoviral vectors provide a preferred system for the production of group C adenoviral vectors (Fallaux et al., 1998).
  • the deletion of E1 sequences provides space for the introduction of foreign genes in the viral vector. Since the maximum size of Ad5 genomes that can be incorporated into virions is limited to about 105% of the wt length, E1-deleted viruses can accommodate approximately 4.8 kb of foreign DNA (Bett et al., 1993).
  • the maximum packaging capacity in virions that lack pIX is reduced to approximately 95% of the normal genome length (Ghosh-Choudhury et al., 1987). This is most likely caused by the reduced stability of pIX- ("pIX-minus") virions.
  • pIX-minus mutant Ad5 can be complemented by episomal expression of pIX in a packaging cell line used for producing viruses (Caravokyri et al., 1995).
  • Ad5 and Ad2 are most commonly used as gene transfer vectors, other serotypes may have preferred characteristics that make them more useful as a therapeutic or prophylactic tool.
  • Subgroup B viruses Ad35 and Ad11 for example are much less prone to neutralisation by human sera than Ad5 and Ad2 viruses (disclosed in WO 00/70071 ).
  • Neutralisation of adenoviral transfer vectors diminishes transduction efficiency in vivo.
  • the infection efficiency of antigen presenting cells, like dendritic cells, by recombinant viruses carrying the fiber of Ad35 was found to be greatly enhanced in vitro compared to Ad5 viruses ( WO 00/70071 , WO 02/24730 ).
  • Ad35-based vectors combine highly improved infection efficiency with low neutralisation in human sera, making such vectors suitable for vaccination purposes.
  • FIG. 1 Map of pWE.Ad35.pIX-rITR ⁇ E3
  • Fig. 2 Gel analysis of PCR fragments generated on Ad35 E1-deleted viruses with and without the E3 region.
  • PI plasmid control
  • M marker: 1 kb plus ladder (Invitrogen);
  • mQ H2O. Indicated genome lenghts are in kb.
  • Fig. 3A Sequence alignments of the proximal pIX upstream sequence regions of various adenoviruses generated with MEGalign software (DNAstar) using Clustal method. Source of sequences are indicated in the text. The Sp1 site and TATA-box in Ad5 and Ad2 (as in Babiss and Vales, 1991) are boxed.
  • Fig. 3B Schematic comparison of putative Sp1 - and TATA-boxes in proximal pIX regions from sequences given in 3A.
  • Fig. 6A Gel analysis of PCR fragments generated on DNA isolated from Ad35.AdApt.Luc and Ad35.AdApt535.Luc viruses or generated on plasmid controls.
  • M Marker (1 kb plus ladder, Invitrogen). Each virus preparation or plasmid control is analysed with two specific PCR amplifications.
  • Fig. 6B Gel analysis of PCR fragments generated on Ad35.AdApt.LacZ ⁇ E3 (35LacZ) and Ad35.AdApt535.LacZ ⁇ E3 (535LacZ) viruses or generated on plasmid controls.
  • FIG. 7 Map of pBr.Ad35. ⁇ SM.AdApt.LacZ
  • Fig. 8 Schematic representation of the putative promoters in the E1B promoter and 55K coding region.
  • Fig. 9 Schematic representation of the restriction sites in the 55K region that can be used to generate distinct fragments for identification of a putative promoter. Numbering of the sites is according to their position in wild type Ad35.
  • Fig. 10 Sequence alignment of the region between the polyA signals of the E1A region and of the E1B/pIX region in three different subgroup B serotypes.
  • FIG. 11 Schematic representation of pBr.Ad35.PRn.
  • FIG. 12 Schematic representation of pBr.Ad35.PRnAE3.
  • Fig. 13 Schematic representation of the system for producing recombinant adenoviral particles in cells such as PER.C6 through a double homologous recombination event.
  • Fig. 14 Alignment of Ad35 and Ad11 pIX-cDNA sequences with wt Ad35 sequence.
  • the sequences obtained from cloned cDNA fragments as described in example 18 were aligned using SeqMan software from DNASTAR.
  • Ad35 cDNA sequences were derived from RNAs isolated from wtAd35- or Ad35E1B+Luc-infected cultures (sequence of one out of seven clones is shown), the Ad11 cDNA sequence from RNA isolated from an wtAd11-infected culture. The sequence numbering is arbitrary.
  • nucleotide 3339 to nucleotide 3628 of the wt Ad35 sequence is shown.
  • the intron sequence (seen as a gap in the cDNA sequences) is flanked by splice donor (SD) and splice acceptor (SA) sites closely matching the known consensus sequences.
  • SD splice donor
  • SA splice acceptor
  • Fig. 16 Transgene PCR results from Ad35 viruses with a 166 bp 3' E1B sequence retained.
  • M 1kb+ marker (Invitrogen)
  • P pAdAptBsuLuc control plasmid.
  • a recombinant group B adenovirus that has a deletion in the E1 region up to the stopcodon of E1B 55K, can accommodate less exogenous sequences than a similar Ad5 recombinant adenovirus. It appears that this is due to a relative underexpression of the pIX gene in said group B virus in a given packaging cell, when the pIX coding region is preceded by sequences between the E1B 55K stopcodon and the pIX startcodon only.
  • viruses can be rendered more stable and/or capable of accommodating more exogenous sequences when either the pIX promoter is at least partly restored by including sequences from the E1B 55K coding region into such a virus, or by using a heterologous promoter to regulate pIX, such that a normal, or even a relative overexpression, of pIX is achieved in a given packaging cell.
  • the present invention provides recombinant subgroup B adenoviruses, nucleic acid that upon introduction into a packaging cell constitutes the genome of such adenoviruses, methods for increasing the stability and/or packaging capacity of such adenoviruses, and packaging cells comprising such adenoviruses, according to the claims.
  • said elevated level of pIX gene product is brought about by retaining or reintroducing part of the E1B 55K sequences in said adenovirus. In other embodiments, said elevated level of pIX gene product is brought about by the expression of the pIX coding sequences under control of a heterologous promoter.
  • the invention further provides a recombinant adenovirus comprising a functional pIX coding sequence under control of an expression sequence, said expression sequence comprising part of an E1B 55K sequence capable of increasing expression of the pIX coding sequence in a given packaging cell, relative to the expression of said pIX coding sequence behind its endogenous proximal pIX upstream sequence without the part of said E1B 55K sequence, with the proviso that said part of an E1B 55K sequence does not code for a functional E1B 55K gene product. It is shown in the present invention that pIX promoter sequences can be present in said E1B 55K sequences, and including these sequences in said expression sequence can therefore increase pIX expression.
  • E I B 55K sequences increases the stability and/or packaging capacity of said recombinant adenovirus, compared to the situation where said pIX coding sequence is behind its endogenous proximal pIX upstream sequence without the part of said E1B 55K sequence.
  • An adenovirus of serotype 35 with a deletion in the E1 region but with an intact E I B 55K coding region has been disclosed in WO 02/40665 .
  • a functional 55K gene product such as present in the disclosed vector, inhibits apoptosis, and hence it is desired to obtain recombinant adenovirus that lacks functional E1B 55K expression, e.g. by mutating the E1B 55K gene or preferably by including only part of the E1B 55K sequences, more preferably including only sequences downstream of the E1B 55K startcodon.
  • said sequences comprise not more than about 680, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 nt of the adenovirus sequences that are directly upstream of the pIX open reading frame.
  • such a recombinant adenoviral vector retains 166 bp of the 3' end of the 55K coding sequence.
  • the invention is not limited to the presence of a sequence that is found in a contiguous stretch directly upstream of the pIX coding sequences in the natural adenovirus. Instead, it will also be possible according to the invention to have sequences that are more upstream, i.e.
  • adenovirus is a subgroup B adenovirus, preferably an Ad35 or Ad11 adenovirus.
  • Adenoviruses of serotypes 35 or 11 have been shown to be particular useful for administration to humans, as there are much less individuals that have neutralizing antibodies to these serotypes than to the serotype 5 hitherto most used ( WO 00/70071 ). It is another aspect of the invention to provide the nucleic acid that can act as the genome of the adenovirus according to the present invention.
  • a modified pIX gene is a pIX gene having a different promoter and/or transcription terminator, and/or mutated coding sequences, e.g. obtained by codon optimisation, introduction of introns that stabilize RNA, and the like.
  • a pIX gene according to the invention comprises genetic information encoding pIX, and includes nucleic acid such as the pIX gene such as found in natural adenoviruses, cDNA and information encoding mutant pIX in the form of allelic variants or nucleic acid encoding mutant pIX that has at least part of the function of pIX which may differ from normal pIX in quantitative or qualitative aspects, derived from pIX by mutation, deletion, addition, or translocation of amino acids, or combinations thereof.
  • a recombinant adenovirus has a deletion of at least the E1B 55K region up to and including the stopcodon of the E1B 55K gene product
  • the pIX open reading frame will be preceded by sequences between the E1B 55K stopcodon and the startcodon of pIX.
  • sequences are herein referred to as the "endogenous proximal pIX upstream sequences" (e.g. the Ad35 pIX upstream sequence in Ad35 based recombinant adenoviral vectors, the Ad11 pIX upstream sequence in Ad11 based recombinant adenoviral vectors, etc.
  • heterologous promoter as used herein is defined as any sequence different from the sequences naturally found upstream of the pIX gene, including the sequences of the E1B 55K region and the endogenous proximal pIX upstream sequences, and being capable of acting as a promoter and thereby regulating transcription of pIX coding sequences.
  • a heterologous promoter may be a sequence at least in part derived from a proximal pIX upstream sequence (i.e.
  • Ad35 derived pIX expression is driven by an Ad5 non-endogenous proximal pIX promoter.
  • any non-endogenous proximal pIX promoter derived from the proximal pIX upstream sequence from a serotype that confers higher levels of pIX expression than the endogenous pIX proximal sequences of the adenoviral vector may be used. Identification of such non-endogenous proximal pIX promoters may be based upon sequence information, such as will be evident to the person skilled in the art from Example 4.
  • the adenoviral vector according to the invention is derived from an adenovirus subgroup E serotype, or preferably of subgroup B serotype.
  • said adenoviral vector is derived from an adenovirus serotype 35 (Ad35), Ad11, Ad7, or Ad4.
  • said non-endogenous proximal pIX promoter is at least in part derived from a proximal pIX upstream sequence of an adenovirus classified in subgroup C, A, D, or F.
  • said non-endogenous proximal pIX promoter is at least in part derived from a proximal pIX upstream sequence of an adenovirus serotype 12 (Ad12), Ad9, or Ad40, or more preferably of Ad5 or Ad2.
  • sequences acting as non-endogenous proximal pIX promoter can be found empirically, by general molecular biology methods known to persons skilled in the art, such as by transcription assays wherein promoters can be routinely tested for strength. It will be clear to the skilled person that elements from a promoter may be swapped without exchanging the whole promoter, e.g. adding, deleting, or mutating known transcription factor binding sequences to a promoter may influence its strength. Mutating at least part of promoter sequences can be done by changing the sequence by mutations, such as by additions, deletions, or exchanging of one or more nucleotides, including stretches of nucleotides with a known function.
  • Substituting promoter sequences is done by replacing part or all of these sequences by a different promoter. Such replacing can be done according to standard molecular biology techniques all well known to the person skilled in the art. Any promoter can be constructed in operable association with the pIX gene of choice, and tested for its effect.
  • heterologous promoter is unrelated to adenoviral non-endogenous proximal pIX promoters. Therefore, heterologous promoters may also be viral promoters, including but not limited to promoters derived from Cytomegalovirus (CMV, e.g. the human CMV immediate early gene promoter, further herein referred to as the CMV promoter), Rous Sarcoma Virus (RSV, e.g. the RSV long terminal repeat promoter, further referred to herein as the RSV promoter), TK, HBV, SV40 and the like.
  • CMV Cytomegalovirus
  • RSV Rous Sarcoma Virus
  • TK e.g. the RSV long terminal repeat promoter
  • HBV SV40
  • adenoviral E1B promoter is used as a heterologous promoter.
  • Cellular promoters can also be used as heterologous promoters, and these include but are not limited promoters from PGK, metallothionein, EF1- ⁇ , ⁇ -actin, and the like. Synthetic or hybrid promoters, comprising elements from more than one promoter, can also be used for the invention, and are all included within the scope of the term heterologous promoter. Promoters used may be constitutive or inducible. In the context of an inducible promoter a promoter is considered suitable for the invention if it gives overexpression in its induced state. Any promoter sequence resulting in overexpression of pIX according to the invention can be used as a heterologous promoter according to the invention.
  • a heterologous promoter may still contain or include part or all of the endogenous proximal pIX upstream sequences, or alternatively wholly replace these sequences, as long as the heterologous promoter according to the invention can cause the overexpression of genetic information encoding pIX in a packaging cell of choice.
  • An elevated or increased level of pIX gene product in the invention is the result of overexpression of the pIX gene in a packaging cell of choice.
  • Overexpression of the pIX gene as used herein is defined as an expression level of pIX, either on RNA or protein level or both, that is higher than the pIX expression level obtained when the coding region of pIX is behind the "endogenous proximal pIX upstream sequences" as defined herein, in a given packaging cell.
  • "Overexpression" is meaningful in the context of the present invention for a particular heterologous promoter-pIX combination of an adenovirus in combination with a particular packaging cell of choice.
  • Methods to determine expression levels are generally well known to persons skilled in the art, and include but are not limited to RT-PCR, Northern blotting, Western blotting, and the like.
  • overexpression would be measured by determining the expression levels in a recombinant adenovirus with a given insert (e.g.
  • luciferase in a given packaging cell of choice, wherein the genetic information encoding pIX is behind the endogenous proximal pIX upstream sequences without E1B 55K sequences, and comparing these expression levels to those in a recombinant adenovirus that is the same except that the pIX coding region is regulated by a heterologous promoter or by sequences from the endogenous E1B 55K gene (its 'natural' promoter that has been at least partly reconstituted).
  • Overexpression of pIX is indicated by a ratio higher than 1 for the expression level obtained by the heterologous or 'natural' promoter over that obtained by the endogenous proximal pIX upstream sequences without E1B 55K sequences.
  • the choice of a packaging cell is determined by factors such as the serotype of the elements of the recombinant adenovirus that interact with the complementing adenoviral functions in the packaging cell, product purity (such as absence of replication competent adenovirus from the generated batch), ease of use, growth characteristics, and the like.
  • packaging cells are known to the person skilled in the art, and include 293 cells, 911 cells, and PER.C6 TM cells as used herein, as well as derivatives thereof adapted for complementation of adenoviral vectors of specific serotypes, such as PER55K.
  • the pIX coding sequences and regulatory sequences driving the expression of pIX can be positioned on their natural location within the adenovirus genome, as well as in different parts of the adenovirus genome, e.g. in the region that originally contained the E3 sequences.
  • Recombinant adenoviruses with increased stability are capable of incorporating larger genomes into virus particles (virions).
  • increasing the stability of recombinant adenoviruses as used herein will allow the recombinant adenoviruses according to the invention to include more foreign genetic information, comprising a gene of interest.
  • recombinant adenoviruses with increased stability according to the invention may be capable of being propagated for more passages without signs of instability. Stability can be measured by several methods known to people skilled in the art, including but not limited to PCR on recombinant virus to demonstrate the presence of desired recombinant adenoviral vectors.
  • a recombinant adenovirus also called recombinant adenoviral vector, or adenoviral vector, as used herein is derived from an adenovirus, lacks at least part of the E 1 region (comprising the E1A and E1B genes) of an adenovirus and can comprise foreign genetic information of which delivery and/or expression by said vector is desired.
  • Exogenous (or foreign) genetic information is any genetic information that is not naturally present in an adenovirus, and is also referred to as transgene. This includes but is not limited to genes of interest, expression cassettes, and the like. Such exogenous genetic information can fill the space in the genome that has become available by the deletion of adenoviral E1 sequences.
  • Recombinant adenoviral vectors are useful for various purposes, such as in gene therapy applications, vaccine preparation, and the like.
  • E3 sequences can be deleted from such adenoviral vectors, to increase the capacity for foreign genetic information in certain preferred embodiments.
  • Other deletions and various combinations of part or complete deletions of E2, E3, and E4 regions combined with the E1 deletion can be used, if necessary in combination with a packaging cell comprising the genetic information lacking in the adenoviral vector, when necessary for replication of the adenoviral vector.
  • All recombinant adenoviruses having a deletion in the E1-region combined with optionally any other deletions in the adenovirus genome are meant to be included within the scope of the present invention.
  • the adenoviruses of the present invention can be used in different settings such as gene therapy or prophylactic and/or therapeutic vaccination, including tumour vaccination and anti-viral vaccination.
  • the adenoviral vector functions as a gene delivery vehicle, wherein a non-native gene is incorporated into the adenoviral genome.
  • the adenoviral particle can subsequently be targeted specifically to target cells of interest; the adenovirus binds to that specific cell either through capsid-receptor binding or through other means, and delivers the transgene.
  • adenoviral vector targeting will be aware of all the different possibilities that are applied to deliver the adenoviral vectors to the cells of interest. Such possibilities include but are not limited to capsid alterations (fiber, hexon and/or penton modifications, such as deletions, swaps between fibers of different serotypes, and additions of peptides and/or other binding moieties), wherein chimeric fibers are produced that recognize a receptor present on the cell of interest, or wherein the binding of the penton-base is utilized.
  • capsid alterations fiber, hexon and/or penton modifications, such as deletions, swaps between fibers of different serotypes, and additions of peptides and/or other binding moieties
  • the present invention also discloses recombinant adenovirus vectors according to the invention, further comprising a sequence encoding a non-adenoviral protein.
  • sequences can be present on different locations within the adenoviral backbone, but preferably they are located in the E1 region, which is lacking in the recombinant adenoviral vectors of the invention.
  • the E1 region is complemented by the complementation elements present in the complementing cells.
  • the direction of the promoter, transgene and other regulatory sequences can be directed towards the left-, as well as to the right inverted terminal repeat.
  • the production of viral vectors based on adenovirus and/or on other viruses such as the Adeno-Associated Virus (AAV), wherein the combination, such as an Ad-AAV chimeric virus, can integrate into the host cell genome is also contemplated.
  • AAV Adeno-Associated Virus
  • Several methods are known in the art for generating integrating adenoviruses.
  • the invention is also useful for the production of adenovirus forms that (specifically, or non-specifically) can integrate.
  • non-adenoviral transgenes can be cloned into the recombinant adenoviral vectors of the present invention. These do not only include regulatory nucleic acid sequences such as enhancers, promoters (e.g. strong non-adenoviral promoters such as the cytomegalovirus promoter, the SV40 promoter and the RSV promoter) and polyadenylation signals, but also heterologous genes for therapeutic purposes.
  • promoters e.g. strong non-adenoviral promoters such as the cytomegalovirus promoter, the SV40 promoter and the RSV promoter
  • polyadenylation signals e.g. strong non-adenoviral promoters such as the cytomegalovirus promoter, the SV40 promoter and the RSV promoter
  • heterologous genes for therapeutic purposes.
  • recombinant adenovirus vectors according to the invention are provided, wherein said non-adenoviral protein is selected from the group consisting of: a cell-death inducing polypeptide, a tumor specific antigen, a viral protein, a hormone and a cytokine.
  • Non-limiting examples of non-adenoviral factors, proteins, polypeptides and peptides are transcription factors, intracellular signalling proteins, phosphatases, kinases, apoptosis inhibiting factors, receptor antagonists, soluble forms of membrane-bound receptors, RNA inhibitors, anti-sense RNA's, decoy factors, ribozymes, and more specifically, thymidine kinase, erythropoietin, novel-erythropoiesis stimulating protein (NESP), IL3, ceNOS, gamma-interferon and gp100.
  • Non-adenoviral viral proteins can be cloned into the recombinant adenoviral vectors provided by the methods and means of the present invention for vaccination purposes.
  • viral proteins include, but are not limited to, gag, pol, env, nef, etc. for HIV vaccines, E6 and E7 proteins for Human Papilloma Virus vaccines, circumsporozoite proteins from Plasmodium protozoa for malaria vaccines, rotavirus components for rotavirus vaccines, ebola proteins for ebola vaccines, the F and G gene products from Respiratory syncytial virus for Respiratory Syncytial virus vaccines, HA and NA for influenza vaccines,etc.
  • Adenoviruses according to the invention are preferably human adenoviruses, i.e. derived from adenovirus that is capable of infecting human cells, but the invention is equally useful for non-human adenoviruses.
  • human adenoviruses i.e. derived from adenovirus that is capable of infecting human cells
  • the invention is equally useful for non-human adenoviruses.
  • a person skilled in the art will be aware of the fact that in addition to all human adenoviruses numerous non-human adenoviruses have been identified in the art. Obviously, also non-human adenoviruses can be applied to reach the same results as disclosed by the present invention.
  • Non-limiting examples of non-human adenoviruses that can be produced using the methods and means of the present invention are canine-, bovine-, monkey- and avian adenoviruses.
  • Serotypes as used herein therefore goes beyond species-restricted serotypes.
  • "Derived from” as used herein means that nucleic acid sequences, genes, or proteins that are normally found in an adenovirus, are used for the generation of recombinant adenoviruses according to the invention. Methods to generate such recombinant adenoviruses are well known to persons skilled in the art, and include but are not limited to general molecular biology methods such as cloning of genetic information into desired constellations by use of restriction enzymes, and the like. "Derived from” is also meant to include the synthetic construction of genetic information based upon knowledge of such genetic information.
  • Such methods include but are not limited to, the use of adenoviral genetic material as a template for PCR to construct a new adenoviral construct that is based upon the sequence of the template adenovirus, the construction of completely synthetic genetic information with a desired sequence e.g. by linking synthetic oligonucleotides to a desired construct, and the like. It is to be understood that 'derived from' does not necessarily mean a direct cloning of the wild type DNA. A person skilled in the art will also be aware of the possibilities of molecular biology to obtain mutant forms of a certain piece of nucleic acid.
  • adenoviruses have been classified into subgroups A-F, which encompass 51 serotypes (see e.g. EP 0978566 ).
  • adenoviral vectors derived from adenoviruses from specific subgroups or from certain serotypes that have a tissue tropism for a desired cell type, e.g. dendritic cells ( WO 02/24730 ).
  • the present invention relates to adenoviral vectors derived from an adenovirus classified in subgroup B.
  • Subgroup B of human adenoviruses comprises Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, and Ad50.
  • Preferred embodiments of the present application relate to recombinant adenoviral vectors derived from Ad35 or Ad11 serotypes. Besides choosing for a serotype for specific applications, so-called chimeric adenoviruses can be used. These comprise parts or all of genetic sequences coding for coat proteins, such as fiber, penton, or hexon, from one or more adenoviral serotypes linked to the remaining genetic information (the 'main' adenoviral vector part) from other serotypes, which can be used to decrease immunogenicity or change the tissue tropism of the 'main' adenoviral vector ( EP 0978566 ).
  • coat proteins such as fiber, penton, or hexon
  • the 'main' part as used herein means that it contributes most of the genetic information to said chimeric virus, and a chimeric adenovirus will therefore be included in the serotype group of the 'main' part of such a virus. It will be clear to those skilled in the art that the present invention can also be used for such chimeric adenoviruses when these could face similar instability problems. It can for instance be expected that a chimeric adenovirus comprising Ad35 sequences as the main part and comprising a fiber that is derived from e.g. an Ad11 adenovirus may have similar instability upon propagation as is reported for the Ad35 recombinant adenoviral vectors described here. Hence, when recombinant adenoviruses are mentioned in this application, chimeric adenoviral vectors are meant to be included in the present invention.
  • a packaging cell and a recombinant adenoviral vector that lack overlapping sequences that would otherwise lead to homologous recombination resulting in replication competent adenovirus ( US patent 5,994,128 ).
  • PER.C6T TM as deposited under no. 96022940 at the European Collection of Animal Cell Cultures at the Center for Applied Microbiology and Research, is therefore a very suitable packaging cell for propagating recombinant adenoviruses.
  • Other methods to decrease the generation of replication competent adenovirus have also been envisaged and concern for instance manipulation of adenoviral sequences to reduce the homology between sequences present in the packaging cell and the vector (e.g.
  • Packaging cells can, besides the obligatory E1 region, comprise other adenoviral sequences to complement other adenoviral functions when these are functionally lacking in the recombinant adenovirus used, such as e.g. parts or all of E2, E4, and the like.
  • the complementing information in packaging cells can be present either integrated in the genome, or as extrachromosomal copies, e.g. on plasmids, vectors, cosmids, and the like.
  • helper viruses which comprise genetic information lacking in the recombinant adenovirus.
  • Recombinant adenoviral vectors are also used as so-called helper viruses used for the production of recombinant adenoviruses that contain a genome deleted for most or all adenoviral genes (gutless vectors or helper dependent adenoviruses).
  • gutless adenoviruses In the final production of such gutless adenoviruses it is necessary to avoid packaging of the helper adenovirus.
  • a person skilled in the art is familiar with the methods to achieve this, for example using a site-specific recombinase on an engineered site in the packaging signal to delete this packaging signal. Often it is necessary to separate remaining helper virus from the desired gutless virus using CsCl-gradient separation.
  • the present invention can equally be applied to increase the stability of the recombinant adenovirus by use of a recombinant helper virus having the increased pIX expression, which can be accomplished by the methods described in the present invention.
  • Any cell containing genetic information that can be used to complement the recombinant adenovirus, to generate recombinant virus particles, is meant to be included in the scope of the meaning of packaging cell. It will be clear to the person skilled in the art that the advantage gained by the present invention is not dependent on the packaging cell used.
  • the genetic information encoding pIX can either be present on the recombinant adenoviral vector but can also be present independent from said recombinant adenoviral vector, and such extraviral genetic information can be present either integrated in the genome, or as extrachromosomal copies, e.g. on plasmids, vectors, cosmids, and the like.
  • Introducing genetic information into a packaging cell can be done according to a variety of methods, such as transfection by lipofectamin, calcium phospate precipitation, viral infection, and the like. Such methods are generally well known to the person skilled in the art, and the method used for introduction of genetic information is not critical for the scope of the invention.
  • Functional pIX in expressible format means genetic information encoding pIX in operable linkage to a promoter or other regulatory sequence capable of driving expression of said genetic information encoding pIX in the packaging cell.
  • Introduction of genetic information into the packaging cell can be done either prior to, concomitantly with, or after the introduction of the recombinant adenoviral vector. It was found that constitutive episomal expression of pIX in a 293-based packaging cell line complements the deficiency of pIX mutant adenovirus type 5 (Caravokyri and Leppard, 1995).
  • adenoviral vectors for such applications special episomal plasmids containing an EBNA1 expression cassette are required, and propagation of adenoviral vectors in such cell lines suffers from the disadvantage that parts of the episome very likely will become part of the recombinant adenoviral vector.
  • genetic information encoding functional pIX is present on the adenoviral vector.
  • the invention demonstrates that a recombinant adenoviral vector derived from Ad35 comprising a pIX gene under the control of an Ad5-derived proximal pIX promoter derived from Ad5 is more stable/can harbour more foreign genetic information than the corresponding virus with the endogenous (i.e. Ad35 derived) proximal pIX upstream sequences.
  • the present invention also provides a recombinant adenovirus comprising a functional pIX coding sequence and having at least a deletion in the E1-region, wherein the pIX coding sequence is under control of a heterologous promoter, and wherein said recombinant adenovirus is derived from an adenovirus other than an adenovirus serotype 5.
  • said heterologous promoter is a non-endogenous proximal pIX promoter.
  • the genetic information encoding pIX is derived from Ad35 or Ad11.
  • a preferred non-endogenous pIX promoter is an Ad5 promoter.
  • the invention provides a recombinant adenoviral vector obtainable by a method according to the invention.
  • recombinant adenoviral vectors are useful, e.g. in the preparation of vaccines ( WO 00/70071 ; WO 01/38362 ; WO 02/24730 ), as gene delivery vehicles, and the like.
  • Choosing a desired main serotype for such recombinant adenoviral vectors can be used for obtaining vectors with an altered tissue tropism as compared to the much-used Ad5 adenoviral vectors, and/or can be used because they are less immunogenic than such Ad5 derived vectors ( WO 00/70071 ).
  • adenoviral vectors For the generation of recombinant adenoviral vectors, it is convenient to clone the transgene into a plasmid (adapter plasmid), containing the left part of an adenovirus lacking E 1 sequences and having restriction enzyme sites for cloning.
  • the recombinant adenoviral vector is then generated by homologous recombination with a cosmid comprising the right part of the adenovirus having at the 5'end overlapping sequences with the 3'end of the adapter plasmid (see examples in present application; method described in WO 99/38362 ).
  • a recombinant nucleic acid sequence comprising an adenoviral left ITR, a packaging signal, other adenoviral sequences with a deletion in the E1 region, at least part of the E1B 55K open reading frame and pIX coding sequences.
  • the invention further provides a recombinant adenovirus packaging cell comprising an recombinant adenovirus according to the invention.
  • said recombinant adenovirus packaging cell comprises a nucleic acid capable of complementing an E1B 55K deficiency of said recombinant adenovirus and wherein said recombinant adenovirus comprises nucleic acid molecule comprising a part of the sequence encoding a E1B 55K gene product increasing the expression of the pIX gene, with the proviso that the latter recombinant nucleic acid molecule does not encode a functional E1B 55K gene product, and wherein said cell and said recombinant adenovirus do not comprise sequence overlap leading to the formation of a recombinant adenovirus comprising a nucleic acid encoding a functional E1B 55K protein.
  • This embodiment is particularly useful for preventing the formation of recombinant adenoviruses
  • Example 1 PER.C6 TM -based complementing cell lines for E1-deleted Ad35 viruses.
  • PER.C6 cells were seeded in 10 cm culture dishes at a density of 3x106 cells/dish in PER.C6 culture medium (DMEM (Gibco BRL) complemented with FBS (Gibco BRL) up to 10% and 10mM MgCl2 (4.9 M stock solution, Sigma)). Two days later, 9 dishes were transfected with 1 ⁇ g ScaI linearised pIG35.55K DNA (described infra) and 9 dishes were transected with 1.5 ⁇ g ScaI linearised pIG35.55K DNA.
  • pcDNA.nlsLacZ (described in WO99/55132 ) is a pcDNA3-based plasmid (Invitrogen) with the nlsLacZ gene driven by the CMV promoter.
  • pcDNA.nlsLacZ also contains a neo r expression cassette.
  • pAdApt35.eGFP an adapter plasmid based on pAdApt35IP1 (described in WO 00/70071 ) but also containing the green fluorescent protein as marker gene, which was cloned into pAdApt35IP1 as HindIII-BamHI fragment derived from pIPspAdapt.eGFP (described in WO 02/24933 )) was digested with PacI to liberate the adenoviral sequences from the plasmid backbone.
  • pWE.Ad35.pIX-rITR (described in WO 00/70071 ) was digested with NotI to liberate the adenoviral sequences from the cosmid backbone.
  • the transfected PER55K(1.0) cultures showed starting CPE (cytopathogenic effect, indicative of virus replication) with approximately 100 events/flask.
  • the untransfected PER55K(1.0) cells were grown confluent with no evidence of CPE.
  • the transfected PER55K(1.0) cultures showed full CPE, with all cells rounded and detached in clumps.
  • the few events of CPE had not progressed and cells were still in monolayer.
  • the clones were seeded at two densities in 6-well plates and one day later infected with 15 ml of the above described crude lysate. CPE was monitored the day after. Of the 146 clones tested in this way 19 gave full CPE at day 2 or 3 and 68 gave full CPE at day 5 or 6. The remaining clones had only partial CPE or showed a few non-progressing events. The latter were indistinguishable from PER.C6 cells that were taken along as a negative control.
  • clones were further screened for the ability to generate recombinant E1-deleted viruses following transfection of the pAdApt35.GFP adapter plasmid and the large pWE.Ad35.pIX-rITR cosmid clone.
  • clones were plated in T25 flasks and transfected with 2 ⁇ g of the adapter and 6 ⁇ g of the backbone plasmid using LipofectAmine as described above. Two days following the transfection, cells were transferred to T80 flasks to prevent overconfluency of the cultures.
  • the amount of virus particles was determined by HPLC (Shabram et al., 1997). Table I presents the yields after downstream processing of medium scale productions of E1- and E1/E3-deleted Ad35 viruses on triple layer flasks with PER55K clone #16 cells. The amount of purified virus particles is comparable with the yields of Ad5-based vectors on PER.C6 cells.
  • Ad35 E1B-55K expression in an Ad5 complementing cell line facilitates replication of Ad35 vectors.
  • the early region-3 of human adenoviruses contains multiple coding regions for proteins that interfere with the host immune response to adenoviral infection. When adenoviral vectors are used as vaccine carrier such interference is unwanted. Therefore, we constructed an Ad35 backbone cosmid lacking the E3 region.
  • construct pBr.Ad35.PRn ( Fig. 11 ; described in example 13 in publication EP1054064 ) was digested with StuI and MluI and the 17.3 kb vector fragment was purified from low melting point (LMP) gel using agarase enzyme (Roche) according to manufacturers instructions. Next, a PCR fragment was generated on pBr.Ad35.PRn using primers 35E3for and 35E3rev.
  • Pwo DNA polymerase (Roche) was used according to manufacturers instructions and program set at: 94°C for 2 minutes, 30 cycles of (94°C for 30 seconds, 58°C for 30 seconds and 72°C for 1 minute) and a final incubation at 68°C for 8 minutes.
  • the 833 bp PCR product was purified using the QIAquick PCR purification kit (Qiagen) and digested with MluI and StuI.
  • the digested DNA was purified from gel using the QIAquick gel extraction kit (Qiagen).
  • the DNA was digested with SwaI and the 22.8 kb vector containing fragment was purified from LMP gel using agarase enzyme as above.
  • Construct pBr.Ad35.PRn ⁇ E3 was digested with PacI and SwaI in the same manner and the 16.6 kb fragment was also isolated using agarase enzyme. Both isolated fragments were ligated using 0.5-0.6 ⁇ g of each frament. Ligated fragments were then packaged using ⁇ -phage packaging extracts (Stratagene) according to manufacturers instructions and mixed with STBL-2 cells. Bacteria were plated on LB+Amp plates and resulting colonies were analyzed for the presence of the correct construct.
  • the right flank of the adapter plasmids and the left end of the backbone plasmid contain homologous sequences mediating recombination events that lead to a complete E1-deleted viral genome (as described in WO 00/70071 ).
  • Transfections were done with 30 ⁇ l Lipofectamine reagent (Invitrogen/Life Technologies) for each set of constructs according to manufacturers instructions. Transfection mixtures were added to PER55K clone 16 cells at 70% confluency in T25 flasks. The following combinations were transfected:
  • Adapter plasmids were digested with pIPsp-1 enzyme to liberate the adenovirus sequences from the plasmid vector backbone.
  • pWE.Ad35.pIX-rITR and pWE.Ad35.pIX-rITR ⁇ E3 were digested with NotI prior to transfection for the same reason.
  • Generation of the adapter plasmids and of the pWE.Ad35.pIX-rITR backbone cosmid is described previously in WO 00/70071 .
  • Generation of pWE.Ad35.pIX-rITR ⁇ E3 is described supra.
  • the mixture was then incubated at 50°C for 1 hour.
  • the viral DNA was isolated using the GeneClean Spin Kit (Bio 101, Inc.). Following elution of the viral DNA in 20 ⁇ l milliQ H2O, the transgene region was analysed by PCR amplification. Hereto, primers AdApt35CMVF and 35pIXR were used. The amplifications were done with 2 ⁇ l of the isolated viral DNA using Taq DNA polymerase (Invitrogen).
  • the reaction mixtures contained 5 ⁇ l 10x buffer (Invitrogen), 2 ⁇ l 50mM MgCl2, 5 ⁇ l 2mM dNTPs, 3 ⁇ l of each primer (10 ⁇ M stock) and 2.5 units Taq enzyme in a total volume of 50 ⁇ l.
  • the program was set at 94°C for 2 minutes followed by 30 cycles of (94°C for 30 seconds, 60°C for 30 seconds and 72°C for 4 minutes). Control reactions were done on 5 ng of adapter plasmids. After completion of the PCR, 5 ⁇ l of the reaction was loaded on gel for analysis.
  • Figure 2 shows the results for the above mentioned transfections.
  • the primers amplify sequences from the 5'end of the CMV promoter to the 5' end of the pIX coding region.
  • viruses without transgene or with GFP insert show the expected band (compare with the plasmid controls; lanes PL for each virus). Smaller fragments are seen with the larger inserts, luciferase and LacZ, and these deletions become more prominent with larger total length of the virus (compare LacZ or Luc viruses with and without E3).
  • increasing genome length corresponds with the occurrence of deletions in the transgene region.
  • the fact that the total genome length also indicated in Fig.
  • Ad35.AdApt.eGFP and Ad35.AdApt.LacZ ⁇ E3, 33.7 and 33.4 kb respectively is comparable while deletions are only found in the LacZ virus sample, indicates that either the sequence or the size of the insert in the former E1 region can also influence the occurrence of the deletions.
  • Example 4 Sequence comparison of the pIX gene region of adenovirus serotype 5 and 35.
  • E1B proteins itself influence the packaging capacity of adenoviruses.
  • E1B-21K protein non-specifically stabilizes transfected DNA (Herrman and Mathews, 1989) and that mutations in the 21 K protein result in degradation of cellular and viral DNA during infection (Pilder et al., 1984; White et al., 1984). Since the Ad5 E1B-21K protein is expressed in PER.C6 cells, these findings do not provide an explanation for our observations.
  • the pIX gene is located directly 3' of the E1B-55K coding region.
  • Ad5 it is known that the pIX promoter and coding sequences are located within the E1B transcription region since pIX and E1B share the poly-adenylation signal.
  • the minimal promoter sequences necessary for pIX expression have been studied in case of Ad5 (Babiss and Vales, 1991). It was shown that a promoter fragment containing the upstream Sp1 site and the TATA-box sequence was sufficient for pIX expression. The spacing between the Sp1 site and the TATA-box as well as the sequence of the TATA-box itself, were shown to influence the level of pIX expression. Whether the corresponding region in Ad35 is also sufficient to drive pIX expression to a level high enough for stable viruses is not known.
  • Sequence comparison revealed that both the Sp1 site and the TATA sequence are different from those found in the Ad5 pIX promoter.
  • sequence information available from Genbank a comparison was made of the proximal pIX upstream sequences (i.e. between the stop codon of E1B-55K and the start codon of the pIX gene) of serotypes from different subgroups.
  • the following adenoviruses with SEQ. ID. NOs.and Genbank reference sequences were used for the comparison: Ad2 (SEQ. ID. NO. 45; Genbank NC_001405), Ad5 (SEQ. ID. NO. 46; Genbank M73260), Ad12 (SEQ. ID. NO.
  • 3A shows an alignment of the above mentioned sequences between the stop codon of the E1B-55K protein (first 3 nucleotides in all sequences) and the start codon of the pIX protein (last 3 nucleotides).
  • the Sp1 site and the TATA sequence in Ad2 and Ad5 are boxed. In most cases there is insufficient homology to directly point the Sp1 and TATA boxes in the other sequences. Therefore, the consensus sequences for GC- and TATA-boxes as published by Bucher, P (1990) was used to identify the putative Sp1 and TATA-box in the various sequences.
  • Fig. 3b shows the putative Sp1 and TATA-box sequences and the spacing between them.
  • Ad12, Ad9 and Ad40 belonging to respectively subgroup A, D and F, have Sp1 and TATA sequences that fairly match the consensus sequence. However, the distance between the two boxes is smaller than for Ad5 and Ad2. This is not unusual since the Ad5 E1B promoter also contains an Sp1 box and a TATA sequence with a spacing of 11 nucleotides. However, a deletion of 9 nucleotides (of the 20) in the Ad5 pIX promoter sequence between the Spl- and TATA-boxes gave reduced pIX levels (Babiss and Vales, 1991).
  • the subgroup B serotypes Ad35, Ad11 and Ad7 as well as subgroup E virus Ad4 have divergent TATA-box sequences and different spacing between the putative Sp1 sequence and the TATA-box.
  • the proximal pIX region in human adenovirus type 4 is identical to that in the simian adenovirus 25 (CV68), a serotype that recently was proposed as therapeutic vector (Farina et al., 2001).
  • CV68 simian adenovirus 25
  • pIX expression may be insufficient for stable capsids.
  • pIX expression is regulated differently in Ad35 viruses and other human and non-human adenoviruses and that regulatory sequences, or even the promoter sequences itself, are located further upstream in the E1B sequences or even more upstream.
  • regulatory sequences or even the promoter sequences itself, are located further upstream in the E1B sequences or even more upstream.
  • the pIX function may be delivered in trans via the packaging cell line.
  • Ad35-based viruses that have a non-endogenous proximal pIX promoter as found in Ad5 viruses, and show that these viruses have a better stability than the unchanged recombinant vectors.
  • pAdApt535 is an Ad35 adapter plasmid having part of the Ad5 pIX promoter sequences but is otherwise identical to Ad35 adapter plasmid pAdApt35IP1 (see WO 00/70071 ). Its construction is described below:
  • a first PCR fragment was generated with primers SV40for and pIX5Rmfe.
  • the reaction was done with Pwo DNA polymerase (Roche) according to manufacturers instructions but with 3% DMSO in the final mix.
  • pAdApt an adapter plasmid for Ad5 E1-deleted viruses (100 ng; see WO 00/70071 ) was taken as template.
  • the program was set as follows: 2 minutes at 94°C and then 30 cycles of (94°C for 30 seconds (melting), 52°C for 30 seconds (annealing) and 72°C for 30 seconds (elongation)) followed by 8 minutes at 72 °C.
  • the resulting PCR fragments contain the 3' end of the SV40 polyadenylation signal from pAdApt and the Ad5 pIX promoter region as present in Genbank Accession number M73260 from nucleotide 3511 to nucleotide 3586 and an MfeI site at the 3'end.
  • a second PCR fragment was generated as described above but with primers pIX35Fmfe and 35R4. 100 ng pAdApt35IP1 was taken as template, the annealing was set at 58°C for 30 seconds and the elongation of the PCR program was set at 72°C for 90 seconds.
  • This PCR amplifies Ad35 sequences from nucleotide 3467 to nucleotide 4669 (sequence numbering as in WO 00/70071 ) and adds an MfeI site to the 5'end.
  • PCR fragments were then digested with MfeI and purified using the Qiagen PCR purification kit (Qiagen) according to manufacturers instructions. Concentration of the purified fragments was estimated by running a sample on agarose gel and approximate equimolar amounts of the two fragments were mixed in a ligation reaction containing 5 ⁇ g DNA, 4 ⁇ l 10x ligase buffer and 2 ⁇ l ligase enzyme (New England Biolabs) in a 40 ⁇ l volume. Following an incubation of > 2 hours at RT, the mixture was loaded on a 1.2% agarose gel in TAE and the DNA fragments of 1.4 kb length were isolated with the Geneclean II kit (Bio 101, Inc.) according to manufacturers instructions.
  • Qiagen Qiagen PCR purification kit
  • the DNA was eluted in 30 ⁇ l sterile H2O and 1 ⁇ l was used in a PCR amplification reaction with primers SV40for and 35R4 as described above.
  • the PCR was done as described above with an annealing temperature of 52°C and an elongation time at 90 seconds.
  • the resulting product was isolated from gel using the Qiagen gel extraction kit and digested with AgeI and BglII.
  • the resulting 0.86 kb band was isolated from gel using the Geneclean II kit according to manufacturers instructions.
  • pAdApt35.Luc (described in WO 00/70071 ) was also digested with BglII and AgeI and the 5.8 kb vector fragment was isolated from gel using the Geneclean II kit as above. This fragment was ligated with the isolated BglII-AgeI fragment described supra containing the Ad5-Ad35 chimeric pIX promoter, to give pAdApt535.Luc ( Fig. 4 ).
  • Example 6 Generation of E1-deleted Ad35-based vectors with adapter plasmids containing the Ad5 pIX promoter.
  • Recombinant viruses were generated by transfection of adapter plasmids and Ad35 vector backbone cosmids on PER55K clone 16 cells as described above.
  • the following set of plasmids were used:
  • AdApter plasmids were digested with PacI except pAdApt535.Luc and pAdApt35.Luc which were digested with pIPsp-1 enzyme and pWE.Ad35.pIX-rITR and pWE.Ad35.pIX-rITR ⁇ E3 were digested with NotI prior to transfection.
  • 2 ⁇ g of each adapter plasmid and 6 ⁇ g of the backbone DNA were mixed with 40 ⁇ l Lipofectamine (Invitrogen/Life Technologies) according to manufacturers instructions and incubated with PER55K clone 16 cells in T25 flasks at 70% confluency.
  • Transfection medium was removed after 4 hrs and cells were further incubated at 37°C/10% CO2. Two days after transfection, cells were passaged to a T80 flask and scored for occurrence of cytopathogenic effect (CPE) the days after. Five days later all cultures showed progressing or full CPE except T6 (no CPE) and T8 (CPE events). Again two days later, T6 and T8 showed starting CPE and all others full CPE. All cultures were harvested by collecting medium and cells. The mixtures were stored at -20°C. Upon thawing of the samples, the mixtures were spun down at 1500 rpm for 15 minutes to pellet cell debris and supernatant were collected.
  • CPE cytopathogenic effect
  • T6-T9 LacZ expressing viruses
  • T6-T9 2 ml was used to infect again PER55K clone 16 cells at 80% confluency in a T80 flask to further amplify the virus titre.
  • Cells and medium were harvested upon progressing (T6+T8) or full CPE (T7+T9) and crude lysates were prepared as described above.
  • the crude lysates obtained in this way were used to isolate viral DNA.
  • 275 ⁇ l of crude lysate material was incubated with 10 ⁇ l 10mg/ml DNaseI at 37°C for 30 minutes.
  • 6.0 ⁇ l 0.5 M EDTA (pH 8.0) 7.5 ⁇ l 20% SDS
  • 1.5 ⁇ l 20 mg/ml Proteinase K was added and mixed by vortexing.
  • the mixture was then incubated at 50°C for 1 hour.
  • the viral DNA was isolated using the GeneClean Spin Kit (Bio 101, Inc.). Viral DNA was eluted in 50 ⁇ l milliQ H2O and 5 ⁇ l samples were used to analyse the transgene region.
  • This PCR specifically amplifies the transgene region in viruses containing the Ad35 pIX promoter.
  • the PCR reaction was done on 5 ⁇ l of the isolated viral DNA samples with recombinant Taq polymerase (Invitrogen) according to manufacturers instructions but with using 4mM MgCl2 and 4 units Taq enzyme in the reactions.
  • the PCR program was set at 94°C for 2 minutes followed by 30 cycles of (94°C for 30 seconds, 60°C for 30 seconds and 72°C for 5 minutes) and ended with a final step of 8 minutes at 68°C.
  • the second was done with the primers AdApt35CMVF and pIX5Rmfe and thus specifically amplifies the transgene region in viruses containing the Ad5 pIX promoter (primer set 2).
  • PCR amplification was done on 5 ⁇ l of the isolated viral DNA using Pwo DNA polymerase (2.5 units/ ⁇ l Genaxis) in 50 ⁇ l volume containing 0.3 ⁇ l of each primer (100 ⁇ M stock), 5 ⁇ l 2mM dNTP mixture, 5 ⁇ l 10x complete buffer (incl. Mg2+), 1.5 ⁇ l DMSO and 0.8 ⁇ l Pwo enzyme.
  • the PCR program was set at 94°C for 2 minutes followed by 30 cycles of (94°C for 30 seconds, 60°C for 30 seconds and 72°C for 5 minutes) and ended with a final step of 8 minutes at 68°C.
  • the heating and cooling ramps were set at 2 °C/second. Then, 5 ⁇ l loading buffer was added to the samples and 8 ⁇ l of the mixture was loaded on gel for analysis.
  • the E1 and E1/E3 deleted Ad35 viruses containing the Ad5 pIX promoter sequence and eGFP transgene had PCR amplified bands at the expected height with no shorter fragments.
  • Fig. 6a shows the results for the PCR amplifications on the E1-deleted Luciferase carrying viruses (transfections T3 and T4).
  • Lanes 5-8 are the control PCRs on AdApt535Luc (lanes 5 and 6) and AdApt35Luc plasmids (lanes 7 and 8) with both primer sets.
  • Lanes 1-4 are PCRs on the viral DNA isolates.
  • Primer set 1 (specific for Ad35 pIX region) amplifies a band of the expected length and shows in addition shorter fragments on Ad35.AdApt35.Luc viruses (lane 4; compare also Fig. 2 Luciferase +E3).
  • primer set 2 (specific for the Ad5 pIX promoter) only shows a band of the expected length with no deletion fragments when viruses are made with the AdApt535.Luc plasmid (lane 1). From this we conclude that the insertion of Ad5 pIX promoter sequences increases the stability and the packaging capacity of Ad35-E1 deleted viruses.
  • Figure 6b confirms these results for Ad35 E1/E3 deleted viruses carrying LacZ as transgene.
  • Lanes 1-4 are the control PCRs on AdApt535.LacZ and AdApt35.LacZ plasmids with each primer set. Some background bands are seen especially with primer set 1 (lanes 2 and 4) but a strong specific band is also seen at the expected height for each primer set on the homologous samples (lanes 1 and 4). Viral DNA was isolated after transfection and after one amplification round as described above. Strikingly, primer set 2 generates the expected fragment on Ad35.AdApt535.LacZ viruses with no deletion fragments (lanes 5 and 9) whereas the sample with viruses containing the Ad35 pIX promoter sequence clearly shows deleted fragments in addition to a fragment of the correct length (visible after amplification (lane 11).
  • the adenovirus insert in the cosmid pWE.Ad35.pIX-rITR contains the Ad35 pIX promoter at its 5' end. This could lead to re-insertion of the Ad35 pIX promoter into viruses generated with the pAdApt535-based adapter plasmids. Therefore, a new version of the Ad35 backbone cosmid is made that lacks pIX promoter sequences. Hereto, a PCR fragment was generated with the pIXcosF-2 and Adapt35-3 primers.
  • the amplification was done with Pwo DNA polymerase (2.5 units/ ⁇ l; Genaxis) in 50 ⁇ l volume containing 3 ⁇ l of each primer (10 ⁇ M stock), 5 ⁇ l 2mM dNTP mixture, 5 ⁇ l 10x complete buffer (incl. Mg2+), 1.5 ⁇ l DMSO, 0.5 ⁇ l Pwo enzyme and 10 ng pAdApt35IP1 template.
  • the PCR program was set at 94°C for 2 minutes followed by 5 cycles of (94°C for 30 seconds, 58°C for 30 seconds and 72°C for 1.5 minutes) and then 25 cycles of (94°C for 30 seconds, 60°C for 30 seconds and 72°C for 1.5 minutes) and ended with a final step of 8 minutes at 68°C.
  • the resulting 1.2 kb PCR product contains Ad35 sequences from nucleotide 3481 to nucleotide 4663 (numbering according to Ad35 sequence as published in WO 00/70071 ) with an AatII and NotI site attached to the 5'end.
  • the PCR product was purified using the PCR purification kit (Qiagen) according to manufacturers instructions and cloned into the pPCR-Script Amp vector (Stratagene) according to manufacturers instructions. The sequence of the cloned fragment is then verified by sequencing and subsequently removed from the construct by digestion with AatII and AgeI. The resulting 780 bp fragment is purified from gel using the Geneclean spin kit (Bio101, Inc.) according to manufacturers instructions.
  • Construct pWE.Ad35 ⁇ NdeI (described infra) is also digested with AatII and AgeI and the resulting 12 kb vector fragment is isolated from gel using the Geneclean spin kit (Bio101, Inc.) according to manufacturers instructions. Ligation of both isolated fragments results in construct pWE.Ad35-3481 ⁇ NdeI.
  • construct pWE.Ad35 ⁇ NdeI is described in WO 00/70071 and contains Ad35 sequences from nucleotide 3401 to the NdeI site at nucleotide 6541 and Ad35 sequences from the NdeI site at nucleotide 33167 to the end of the right ITR whereby both Ad35 fragments are linked via the NdeI site (see also Figure 13 in WO 00/70071 ).
  • pWE.Ad35-3481 ⁇ NdeI is then linearised with NdeI, dephosphorylated with CIP enzyme (New England Biolabs) and purified from gel using the Geneclean spin kit (Bio101, Inc.) according to manufacturers instructions.
  • This vector fragment is then ligated to a 26.6 kb NdeI fragment isolated from Ad35 wt DNA after which the mixture is used to package the cosmid using ⁇ -phage packaging extracts (Stratagene) according to manufacturers instructions.
  • the resulting mixture is used to transform STBL-2 bacteria (Invitrogen), giving rise to pWE.Ad35-3481.
  • Construct pIG35.55K contains the coding sequences of the Ad35 E1B-55K gene operatively linked to the human phosphoglycerate kinase promoter (hPGK) and the HBV poly-adenylation sequence. In addition, it contains the neomycine resistence gene operatively linked to the RSV promoter and HBV pA. The construction of pIG35.55K is described below.
  • Construct pIG270 (described in WO 00/70071 ) was digested with EcoRI, treated with Klenow enzyme and purified using a PCR purification kit (Qiagen) according to the manufacturers instructions. The recovered DNA was then digested with AgeI and the ⁇ 5 kb vector fragment was isolated from gel using the Geneclean kit (Bio101, Inc.) according to manufacturers instructions. Next, Ad35 E1B-55K sequences were amplified by PCR on pIG270 template DNA using the 35D21 and 35B3 primers. The PCR amplification was done with Pwo DNA polymerase (Roche) on 2 ng template DNA according to manufacturers instructions but with using DMSO at a final concentration of 3% in the PCR mixture.
  • Pwo DNA polymerase Roche
  • the program was set at: 94°C for 2 minutes followed by 25 cycles of (94°C for 30 seconds, 56°C for 30 seconds and 72°C for 30 seconds) and ended by a final incubation of 72 °C for 10 minutes.
  • the resulting PCR fragment was purified using the PCR purification kit (Qiagen) and digested with NcoI. Following Klenow treatment to fill in the protruding ends, the DNA was further digested with AgeI and again column purified. The thus treated PCR fragment was then cloned into the above prepared EcoRI/AgeI digested vector fragment to give pIG270. ⁇ E1A ⁇ 21K.
  • pIG270. ⁇ E1A ⁇ 21K was digested with AvrII and XbaI and protruding ends were filled in using Klenow enzyme.
  • the 2.9 kb fragment containing the PGK promoter and Ad35 E1B-55K sequences was isolated from gel as described above.
  • pRSVneo4 construction described infra
  • BglII blunted with Klenow enzyme, dephosphorylated and isolated from gel.
  • the blunted AvrII/XbaI fragment from pIG270. ⁇ E1A ⁇ 21K was then ligated into the above prepared pRSVneo4 vector fragment to give pIG35.55K.
  • pRSVneo4 was generated as follows: Construct pRSVhbvNeo (described in WO 00/70071 ) was digested with ScaI and BamHI and protruding ends were filled in using Klenow enzyme. The 1070 bp fragment containing part of the Ampicilin gene and the RSV promoter was isolated from gel using the Geneclean kit (BIO 101, Inc.). Next, pRSVhbvNeo was digested with ScaI and EcoRI, blunted with Klenow and the 3.2 kb fragment containing the neo gene, HBVpA, vector and part of the Ampicilin gene was isolated as above. The two fragments were then ligated to give pRSVneo4.
  • Example 9 Increased pIX expression mediated bv the RSV promoter increases stability of Ad35 viruses.
  • the RSV promoter was inserted into the Ad35 adapter plasmids containing the LacZ or Luciferase reporter gene.
  • the RSV promoter corresponds to an NruI/ApaLI fragment obtainable from pRc-RSV (Invitrogen). Protruding ends were filled in using Klenow enzyme (New England Biolabs) according to manufacturers instructions.
  • the 388 bp fragment containing the RSV promoter was isolated from agarose gel using the QIAquick Gel Extraction kit (Qiagen).
  • Adapter plasmids pAdApt35.Luc and pAdApt35.LacZ were linearized with BglII followed by Klenow treatment to blunt the ends. BglII digests just behind the SV40 poly-adenylation sequence of the transgene expression cassette.
  • BglII digests just behind the SV40 poly-adenylation sequence of the transgene expression cassette.
  • the treated adapter plasmids were then dephosphorylated using Shrink Alkaline Phosphatase (SAP) according to manufacturers (Roche) instructions.
  • SAP Shrink Alkaline Phosphatase
  • the isolated RSV promoter fragment was then ligated with each of the treated vectors and transformed into DH5 ⁇ competent bacteria (Invitrogen). Colonies were analyzed for forward oriented insertion of the RSV promoter relative to the pIX gene resulting in pAdApt35.Luc.rsv and pAdApt
  • an adapter plasmid was generated from sequences that were isolated by PCR from an Ad35 recombinant virus that resulted after deletion of the transgene region.
  • Analysis of a crude lysate preparation resulting from a transfection of pAdApt35.LacZ and the pWE.Ad35.pIX-rITR Ad35 backbone constructs and subsequent plaque purification showed that the virus had a deletion in the transgene region of approximately 2.8 kb.
  • the 5' sequences from this virus were PCR amplified from isolated DNA using primers 35F1 and 35R4. The reaction was performed with Pwo polymerase (Roche) according to manufacturers instructions.
  • Program settings were as follows: 94°C for 2' then 5 cycles of (94°C for 30 sec.; 48°C for 30 sec.; 72°C for 2.5 min.) followed by 25 cycles of (94°C for 30 sec.; 56°C for 30 sec.; 72°C for 2.5 min.), and ended by 8 min. at 68°C.
  • the resulting 2 kb fragment was purified by the PCR purification kit (Qiagen) and cloned into the pCR-Script-Amp vector (Stratagene) according to manufacturers instructions resulting in pCR.Ad3502.8kb.
  • This plasmid was sequenced to determine the extent of the deletion. The deletion affected most of the CMV promoter, the transgene and SV40 polyA. This resulted in linking of the 5' 317 bp of the CMV promoter to the Ad35 sequences upstream of the pIX gene.
  • This CMV fragment contains three GC-boxes and a 21-bp repeat (Boshart et al., 1985).
  • the pCR-Script-based vector containing the amplified sequences (renamed in pCR.C4) had a unique AvrII site preceding the ⁇ CMV-pIX sequences.
  • the vector was linearized with AvrII, blunted with Klenow enzyme and dephosphorylated using SAP enzyme (Roche) as described above.
  • Adapter plasmids pAdApt535.LacZ (Example 5) and pAdApt.Luc ( WO 00/70071 ) were digested with AvrII and BglII and DNA was treated with Klenow to fill protruding ends.
  • the fragments corresponding to LacZ and Luciferase expression cassettes (CMV-TG-pA) were isolated from gel as above and ligated with the AvrII linearized pCR.C4 vector. Transformation in competent cells as above and selection of colonies that had the cassettes in the forward orientation relative to the left ITR, resulted in pCR.C4.LacZ and pCR.C4.Luc.
  • Ad35 viruses were generated as described in example 6 using the new adapter plasmids: pCR.C4.LacZ digested with PacI, pCR.C4.Luc digested with ApaI and
  • pAdApt35.LacZ.rsv and pAdApt35.Luc.rsv each digested with PI-PspI.
  • the adapter plasmids were cotransfected onto PER55K cells ( WO 02/40665 ) with pWE.Ad35.pIX-rITR or pWE.Ad35.pIX-rITR ⁇ E3 digested with NotI.
  • adapter plasmid pBr.Ad35. ⁇ E1A ⁇ 21K.Luc (construction described below) was digested with PI-PspI and cotransfected with pWE.Ad35.pIX-rITR digested with NotI.
  • CPE cyto-pathogenic effect
  • A549 cells were seeded in 6-well plates at 5x105 cells/well and after 5hours infected with 10, 1 or 0.1 ⁇ l of each of the LacZ virus stocks and incubated for two days. A549 cells were then stained for LacZ activity and blue cells were counted. The percentage of blue cells is given in Table II.
  • LacZ expressing viruses are clearly more stable when the RSV promoter is driving expression of the pIX gene as compared to the deleted CMV promoter. These results are confirmed with the Luciferase viruses.
  • A549 cells were seeded in 24-well plates at 1x105 cells/well and infected with 10, 1, 0.1, or 0,01 ⁇ l of the virus stocks and incubated. After two days, cells were washed with PBS twice and resuspended in 100 ⁇ l lysis buffer (Promega) and stored at -20°C until use. Luciferase was measured using the Steady-Glo luciferase assay system (Promega) according to manufacturers instructions. Results are presented in Table III.
  • PER55K cells were seeded in 6-well plates at 0.9x106 cells/well and infected with different 10-fold dilutions of the Ad35.AdApt.LacZ crude lysates. Dilutions from 10-5 to 10-8 were plated and the next day an agar overlay was added.
  • cells were first washed with PBS and then 3 ml of a pre-warmed agar solution prepared by mixing 2xMEM (GibcoBRL; 9.14 ml), FBS (Gibco; 0.36 ml), MgCl (4,9 M; 0.037 ml) with agarose (SeaPlaque GTG; 2.5% in H2O, 7.2 ml), was added. After solidification plates were further incubated at 37°C/ 5% CO2. Four days later plaques were visible and LacZ staining solution was added to the wells on top of the agar and allowed to drain.
  • 2xMEM GibcoBRL; 9.14 ml
  • FBS Gibco
  • MgCl 4,9 M; 0.037 ml
  • agarose SeaPlaque GTG; 2.5% in H2O, 7.2 ml
  • This invention for the first time provides a stable recombinant adenovirus derived from or based upon an adenovirus serotype 35 lacking expression of a functional E1B gene. Such an adenovirus has at least a deletion in the E1-region.
  • said stable recombinant adenoviruses have foreign insert sequences of more than 4.2 kb, and a packaged genome size of more than 33.4 kb, using methods according to the invention. It is therefore an object of the present invention to provide a stable recombinant adenovirus that: a) harbours a foreign nucleic acid sequence of more than 4.2 kb, and/or b) has a packaged genome size of more than 33.4 kb.
  • the invention provides an Ad35 or Ad11 based recombinant adenovirus that: a) harbours a foreign nucleic acid sequence of at least 4.6 kb, and/or b) has a packaged genome size of more than 33.8 kb. Alternatively or in addition thereto, said packaged genome sizes are at least 34.6, 35.0, 36.1 and 36.5 kb, respectively.
  • the foreign sequences in these embodiments may include a heterologous promoter driving expression of pIX.
  • said stable recombinant adenovirus is of serotype 11.
  • a stable adenovirus according to this aspect of the invention can be passaged on a packaging cell to provide a batch of the recombinant adenoviruses, with less than 10%, preferably less than 5%, preferably none of separate clones giving rise to deletions in the foreign sequences in the recombinant adenovirus, as can be measured e.g. by the PCR method exemplified in Example 3.
  • pBr.Ad35. ⁇ E1A ⁇ 21K.Luc was made as follows. Construct pBr.Ad35. ⁇ E1A ⁇ 21K ( WO 02/40665 ) was digested with HpaI, dephosphorylated with CIP (New England Biolabs) and the 5 kb vector fragment was isolated from gel. Construct pBr.Ad35. ⁇ E1A.Luc was also digested with HpaI and the 3.3 kb insert was isolated from gel and ligated with the isolated vector fragment. Following transformation into competent STBL-2 cells (Invitrogen), a colony was selected with the insert in the correct orientation. This gave construct pBr.Ad35. ⁇ E1A ⁇ 21K.Luc.
  • pBr.Ad35. ⁇ E1A.Luc (also called pBr.Ad35.E1B+.Luc, because it still contains the E1B region) was made by inserting the AdApt.Luc cassette, taken from pAdApt.Luc after AvrII and BglII digestion and blunting with Klenow enzyme, into the vector fragment pBr.Ad35.leftITR-pIX ( WO 02/40665 ) digested with SnaBI and HindIII and blunted with Klenow. Colonies with the expression cassette in the forward orientation were selected, giving pBr.Ad35. ⁇ E1A.Luc.
  • Ad35 recombinant viruses in which the coding regions for E1A and E1B are completely removed become progressively more unstable if the genome size is increased.
  • addition of a heterologous promoter driving pIX expression can overcome the instability.
  • Ad35 viruses that retain the complete E1B-55K coding sequence can be produced on PER.C6 and are stable.
  • viruses that retain the full E1B coding sequence WO 00/70071 ; Abrahamsen et al., 1997).
  • pBr.Ad35 ⁇ SM.AdAptLacZ is first generated as follows. Construct pBr.Ad35.1ITR-pIX (described in WO 00/70071 ) is digested with SnaBI and MfeI, blunted with Klenow and de-phosphorylated with SAP enzyme (Roche). The 4.4 kb vector fragment is then isolated from agarose gel.
  • pAdApt.LacZ Ad5-based adapter plasmid pAdApt with LacZ transgene insert; WO 99/55132 ) is digested with AvrII and BglII (and, optionally, SalI to increase the difference in fragment size) and blunted with Klenow enzyme.
  • the 4.2 kb CMV.LacZ.pA insert is then isolated from gel. Both isolated fragments are then ligated to give pBr.Ad35 ⁇ SM.AdAptLacZ ( Figure7 ).
  • the orientation can be checked by restriction digestion since ligation in the correct orientation restores both the AvrII site and the MfeI site.
  • Additional constructs are made by using enzymes DraIII, Bsu36I, BssHII or BamHI, and digest the vector partially (the LacZ gene also contains a recognition site for these enzymes) using methods known in the art, followed by selection of the correct clone. The stability is tested as described above for the Ad35.AdApt.LacZ.rsv construct. Constructs that are stable (i.e. do not acquire deletions in the transgene region) contain proper regulatory regions for pIX expression. In addition, it is possible to directly test promoter activity in a given sequence by inserting the sequence upstream of a reporter gene. pGL3basic (Promega) is such a reporter gene construct.
  • the region between MunI and the start of the pIX gene was amplified using primer set Ad3555KmfeF and Ad35pIXNcoR. This PCR (2 minutes 94°C; then 30 cycles of [30 seconds 94 °C, 30 seconds 59°C, 60 seconds 72°C]; followed by 8 minutes 68°C; enzyme: Pwo (Genexis) according to manufacturer's instructions, with additional 3% DMSO) amplified Ad35 sequences from 2804 to 3491 (numbering as in wt Ad35) thereby changing the sequence around the start codon of pIX into an NcoI site and introducing an HindIII site at the 5' end.
  • This amplified fragment is digested with HindIII and NcoI and cloned into pGL3basic digested with the same enzymes generating pGL3-MN.
  • pGL3-MN is than used to delete sequences upstream of the Luciferase coding region by combining HindIII digestion with e.g. PacI, NsiI, StuI, Bsu36I, BssHII or BglII, followed by blunting of the protruding ends and religation.
  • Promoter activity is tested by transient transfection of the obtained constructs into PER.C6 cells using lipofectamine reagent according to manufacturers instructions.
  • Luciferase activity is analysed two days after transfection using Steady-Glo luciferase assay system (Promega) according to manufacturers instructions.
  • different regions are inserted, which regions are generated by PCR amplification using a 5'(forward) primer directed to a specific sequence in the Ad35 E1B-55K region and having a HindIII site attached at the 5' end combined with the Nco-pIXrev primer. In this way one is not limited to the presence of a unique restriction site for cloning.
  • Figure 8 shows the promoter scores (minimum set at 0.65) for the E1B promoter directly linked to the 55K coding sequence (as in pBr.Ad35. ⁇ E1A ⁇ 21K).
  • the regions marked A corresponds to the E1B promoter and regions B and C locate within the 55K coding region.
  • the pIX upstream region is not recognized as a promoter sequence.
  • Region C has the highest score (0.96) of the three (even higher than the known E1B promoter) and may therefore comprise sequences that influence pIX expression.
  • Figure 9 schematically depicts these fragments and their location relative to the putative promoter region and the pIX gene.
  • the fragments are generated by restriction digestion using the indicated enzymes. Fragments are blunted with Klenow (5' protruding ends) or T4 DNA polymerase (3' protruding ends) and cloned into the NcoI site of the pGL3basic vector (Promega) also blunted by Klenow treatment and de-phosphorylated by SAP treatment (Roche). Following transformation into competent bacteria obtained plasmids are checked for the orientation of the insert by restriction digestion.
  • Promoter activity is then analysed by transient transfection of the obtained luciferase constructs into PER.C6 cells using lipofectamine reagent according to manufacturers instructions.
  • Empty pGL3basic plasmid serves as a negative control. Additional controls are made by cloning i) a BglII-MfeI fragment from pAdApt535 containing the Ad5 pIX promoter, ii) a 388 bp NruI-ApaLI RSV promoter fragment (described above), or iii) the Ad35 pIX upstream region as a PCR fragment into the blunted NcoI site of pGL3basic as described above.
  • Ad35 upstream pIX region is amplified on pAdApt35IP1 using primers SV40-for and 5'-phosphorylated Ad35pIXrev. Following amplification the DNA is digested with BglII and treated with Klenow.
  • Constructs are also transfected into human cells not containing adenovirus E1 (e.g. A549, Hela) to investigate the dependency on E1A expression.
  • E1 e.g. A549, Hela
  • the fragments are also cloned into an adapter plasmid pBr.Ad35 ⁇ SM.AdAptLacZ (see above) to be able to generate recombinant viruses and study viral genome stability.
  • construct pBr.Ad35 ⁇ SM.AdAptLacZ is digested with MfeI and BglII, blunted with Klenow enzyme and dephosphorylated.
  • DNA can be ligated with the fragments described above (see Fig. 9 ) to give rise to a set of adapter plasmids that have varying lengths of 55K fragments upstream of the pIX gene.
  • Viruses can be generated with the construct pWE.Ad35.pIX-rITR as described above. Control transfections are done with the pBr.Ad35 ⁇ SM.AdAptLacZ, pAdApt35.LacZ and pAdApt35.LacZ.rsv constructs. Upon appearance of full CPE, cells and medium are harvested by one freeze/thaw cycle and used to re-infect fresh PER55K cells. Cells and medium are again harvested at full CPE, crude lysates are prepared and used to perform a plaque assay. After appearance of plaques, X-gal staining solution is added to check for LacZ expression.
  • pIX gene expression may be driven by the E1B promoter as a heterologous promoter for the generation of recombinant viruses.
  • pAdApt535.LacZ is digested with BglII and MfeI followed by Klenow treatment to blunt ends and dephosphorylation.
  • the thus treated 4.8 kb vector fragment is then isolated from gel.
  • the E1B promoter region is isolated as a PCR fragment using pBr.Ad35.leftITR-pIX as target DNA and the Epr-F and Epr-R primers, whereby both primers are phosphorylated.
  • This plasmid is then used to generate Ad35-based viruses and test stability as described before.
  • RNA is isolated from infected cells and pIX containing RNAs are identified by hybridisation with a labelled specific probe.
  • PER55K cells are infected at a multiplicity of infection of 10 and 50 with the following viruses: wtAd35, Ad35.E1B.AdApt.Luc, Ad35 ⁇ E3.AdApt.Luc, Ad35 ⁇ E3.AdApt535.Luc, Ad35.AdApt.Luc.rsv.
  • Infected cells are harvested after 8 hrs (wtAd35 also after 2 and 18 hrs) and RNA is isolated using TRI-zol Reagent (Invitrogen).
  • RNA is size fractionated on an 1.5% agarose gel, transferred to a Northern blot and hybridised to a 32P-labeled probe derived from the pIX coding region. Procedures are known in the art (described in Molecular Cloning: A laboratory manual, by Sambrook and Russell, 2001 or earlier versions). The length of the RNA can be determined if known RNA size markers are included and will give an indication of the RNA species that contain pIX sequences. To identify the mRNAs that start in the E1B promoter, the blot can be stripped and re-hybridised with a 5' 21K probe. pIX-containing transcripts that do not hybridise to the E1B-21K probe are likely generated by a promoter different from the E1B promoter.
  • RNA is reverse transcribed into cDNA and the cDNA is used to specifically amplify 5' ends of pIX containing RNAs using the GeneRacer System (Invitrogen) according to manufacturers instructions with the reverse primers directed to pIX coding sequences: pIXrev and the nested primer pIXrev-N2.
  • Cloning and sequencing of the amplified fragments gives the location of the transcription start sites and 5' sequences of mRNAs that contain pIX sequences. In this way the possible pIX coding mRNAs are identified. The correlation between the levels of pIX expression and stability of the corresponding recombinant adenoviruses can thus also be determined.
  • Example 11 E1B and pIX sequences from group B adenoviruses.
  • Ad35 virus As an example.
  • Other members of the subgroup B that have considerable homology to each other are could have comparable pIX regulation.
  • Ad7 (SEQ. ID. NO. 57) is available via Genbank Accession number X03000.
  • the sequence of Ad11 (SEQ. ID. NO. 56) was revealed by shotgun sequencing of DNA isolated from Ad11p wt viruses performed by Lark Technologies (UK) similar as described ( WO 00/70071 ) for the Ad35 sequence (SEQ. ID. NO. 55).
  • Ad11 and Ad35 are highly homologous to each other (overall 98.1% similarity), and the main differences are located in hexon and fiber knob.
  • the Ad11 sequence is also disclosed in W0 02/053759 .
  • Ad35 has an overall similarity (in this region) of 98.4% to Ad11 and 82.9 % to Ad7. This makes it very likely that pIX expression is regulated in the same way in these viruses.
  • the methods and means according to the invention as exemplified in the previous examples can be used accordingly to increase the stability and/or insert capacity of other recombinant adenoviruses of subgroup B, herein exemplified by Ad11 and Ad7.
  • Example 17 E1-deleted Ad35 viruses with a heterologous promoter driving pIX expression.
  • pAdApt35LacZ, pAdApt35.LacZ.rsv (example 9), pAdApt535.LacZ (example 5) and pAdApt35BLacZ (containing the Ad35 E1B promoter sequence in front of the pIX gene; described below) were digested with pIPsp-1 and used to generate viruses with NotI digested cosmid pWE/Ad35-3481 and pWE/Ad35-3481 ⁇ E3 (example 7) as described in example 2 (and in WO 00/70071 ).
  • viruses were generated with adapter plasmid pBr.Ad35 ⁇ SM.AdAptLacZ ( Figure 7 ; example 10).
  • This adapter plasmid is deleted for E1A and a large part of the E1B sequences. It retains 0,6 kb of the 3' E1B-55K sequence and also has wt sequences between the stop codon of 55K and the start codon of pIX.
  • the cells and medium were harvested, freeze/thawed and centrifuged to remove the cell debris. The supernatant (cleared lysates) of each of the transfections was then used to perform a plaque assay as described in example 9.
  • Ad35.AdApt.LacZ.rsv (105%) are also at the border of the theoretical packagable size. Altogether the results show that a heterologous promoter driving pIX expression improves the maximum tolerated packaging size and the stability stability of E1-deleted Ad35 viruses. The same is true for viruses that have a longer endogenous proximal sequence (Ad35ASM.LacZ) suggesting that the additional (E1B 55K) sequences herein contain regulatory elements for pIX expression.
  • pAdapt35BLacZ is an Ad35 adapter plasmid with the Ad35 E1B promoter sequence regulating the pIX gene.
  • Adapter plasmid pAdApt35BLacZ was generated as follows: The E1B promoter fragment was amplified using the primers 35E1Blong and Ad35E1bpromrev. Both primers were phosphorylated. The reaction was done with Pwo DNA polymerase (Inno-train, Diagnostic GmbH) according to manufacturer instructions. pBr.Ad35.leftITR-pIX was used as template DNA, (25 ng, described in WO 02/40665 ). The program was set as follows: 2 minutes at 94°C and then 30 cycles of (94°C for 30 seconds, 60°C for 30 seconds and 72°C for 1 minute) and ended by 10 minutes at 72°C.
  • the cooling/heating slope was set at 2°C/sec. This PCR results in amplification of the potential E1B promoter of Ad35 of 125 nucleotides.
  • Construct pAdapt535.LacZ (example 5) was then digested with MfeI and BgIII. After digestion the vector was treated with Klenow enzyme to create blunt ends. A dephosphorylation step was done using SAP (Roche). The thus treated 8 kb vector fragment was then isolated from gel. The E1B promoter region was also isolated from gel. These two fragments were ligated and transformed into DH5 ⁇ -T1r competent cells (Invitrogen). The correct orientation of the E1B promoter in the resulting plasmid was confirmed by digesting with HpaI and ApaLI. After selection of the correct clone the inserted E1B promoter sequence was also verified by sequencing.
  • Example 18 The promoter of the pIX gene is located in the 3' end of the E1B-55K coding sequence in Ad35 and Ad11 viruses.
  • pIX promoter in subgroup B viruses would be located in the E1B-55K coding region.
  • pIX mRNA cap site as described in example 10.
  • wtAd35, wtAd11 and Ad35.E1B + .AdApt.Luc viruses were used to infect PER55K clone 16 cells at an MOI of 50 VP/cell.
  • wtAd5 was taken along since the promoter and mRNA start site of this virus is known.
  • RNA was isolated from the infected cultures at 16-18 hrs post-infection using TRIzol agent (Invitrogen) as described by the manufacturer.
  • RNA was stored in 100% formamide.
  • the GeneRacer Kit (Invitrogen) was used to amplify the 5' end of pIX transcripts, in order to locate the start of transcription.
  • 5 ⁇ g RNA was purified from the formamide by sodium acetate precipitation as described in the GeneRacer protocol. Purified RNA was treated according to manufacturer's Protocol for amplification of the 5' end of the pIX mRNA. After phosphatase treatment and subsequent removal of the cap structure with tobacco acid pyrophosphatase and ligation of the Generacer RNA oligo, SuperScript TM II Reverse Transcriptase from the kit was used for cDNA synthesis.
  • cDNA was synthesized by reverse transcription using a gene specific (reverse) primer for pIX.
  • Ad35 wt and E1B+Luc virus
  • wtAd11 primer pIXrev was used
  • Ad5 the primer pIXrev-Ad5 was used.
  • the resulting cDNA (1 ⁇ l of unknown concentration) was used as a template for PCR to generate dsDNA. This PCR was done using Pwo DNA polymerase (Roche) according to manufacturers instructions, and with addition of DMSO (Sigma; 3% v/v).
  • the amplification was done with the GeneRacer 5' primer from the kit which is specific for the oligonucleotide ligated to the 5' end of the mRNA, and the gene specific reverse primers, as mentioned above. Reaction conditions were as follows: denaturation at 94°C for 2 minutes, followed by 30 cycles of (94°C for 30 seconds, 64°C for 30 seconds, 72°C for 2 minutes) and finished by elongation at 68°C for 8 minutes. The resulting DNA fragments were size separated by electrophoresis on a 1.0% agarose gel. For Ad5 a 480 bp fragment and for Ad35 (both viruses) and Ad11 a 200 bp fragment was obtained. Ad11 also showed a 2 kb fragment.
  • Ad35 sequence SEQ. ID. NO. 60. The alignment reveals the location of a spliced intron sequence in the pIX mRNA. In Fig. 15 the location of the identified cap site and splice sites is schematically shown in the E1B-pIX region of Ad35.
  • Ad5 the expected pIX mRNA (Babiss and Vales, 1991) was identified with the cap site located at position 3580 (not shown; numbering as in genbank accession no. M73260).
  • Ad35 the cap site was located in the 3' end of the E1H-55K gene at position 3339 on an A residue (Ad35 sequence WO 00/70071 ).
  • Ad11 the cap site was similarly found on a T residue (position 3339 in Genbank Acc No. AY163756).
  • sequence between the stop codon of the 55K gene and the start codon of the pIX, where in Ad5 viruses the promoter for pIX is located is spliced out of the mRNA in Ad35 and Ad11 viruses.
  • Example 19 E1-deleted Ad35 viruses that retain a short stretch of 3' E1B-55K sequence have a larger packaging capacity.
  • Ad35 adapter plasmid that retains 166 bp of the 3'end of the 55K coding sequence (pAdApt35Bsu.Luc) and the generation of an E1-deleted Ad35Luc virus with increased stability and/or packaging capacity.
  • This 166 bp sequence does not code a functional 55K gene product but contains the pIX mRNA cap site identified in the previous example in its natural position relative to the pIX coding sequence.
  • Construct pAdApt35Bsu.Luc was generated as follows:
  • Table I Yields of E1- and E1/E3- deleted Ad35 viruses on clone #16 cells produced on triple layer flasks.
  • Virus Scale T175 111 flasks
  • Table II Transgene (LacZ) activity test on A549 using crude lysates from second passage virus.
  • % blue cells is given for each amount of virus used for infection.
  • Table III Transgene (Luciferase) activity test on A549 using crude lysates from second passage virus. Activity is expressed in relative light units (RLU).
  • Ad35.AdApt.LacZ 50% 36,1 kb Ad535.AdApt.LacZ NP 100% 36,1 kb Ad35.AdAptB.LacZ 5% 80% 36,2 kb Ad35.AdApt.LacZ.rsv 90% 100% 36,5 kb Ad35 ⁇ SM.LacZ 50% 100% 36,7 kb

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EP03753569A 2002-04-25 2003-04-24 Stable adenoviral vectors and methods for propagation thereof Expired - Lifetime EP1497440B1 (en)

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EP03753569A EP1497440B1 (en) 2002-04-25 2003-04-24 Stable adenoviral vectors and methods for propagation thereof
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AU2003271738C1 (en) 2008-04-17
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CA2478508A1 (en) 2003-12-31
PL373337A1 (en) 2005-08-22
CA2478508C (en) 2013-07-02
KR20040106365A (ko) 2004-12-17
JP4495588B2 (ja) 2010-07-07
NZ534865A (en) 2008-07-31
KR101006594B1 (ko) 2011-01-07
US7285265B2 (en) 2007-10-23
HK1069185A1 (en) 2005-05-13
JP2005523730A (ja) 2005-08-11
WO2004001032A3 (en) 2004-04-01
ATE405663T1 (de) 2008-09-15
WO2004001032A2 (en) 2003-12-31
CN100374574C (zh) 2008-03-12
US8052967B2 (en) 2011-11-08
DK1497440T3 (da) 2008-12-01
AU2003271738A1 (en) 2004-01-06
SI1497440T1 (sl) 2009-02-28
US20050163753A1 (en) 2005-07-28
ES2310247T3 (es) 2009-01-01
IL164802A0 (en) 2005-12-18
NO334299B1 (no) 2014-02-03
PL208588B1 (pl) 2011-05-31
AU2003271738B2 (en) 2007-10-11
EP1497440A2 (en) 2005-01-19
NO20045131L (no) 2005-01-21
CN1650021A (zh) 2005-08-03

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